High Prevalence of Ciprofloxacin Resistance in Community Associated Staphylococcus aureus in a Tertiary Care Indian Hospital

We have studied the nature of ciprofloxacin resistance in methicillin sensitive and resistant Staphylococcus aureus among patients in a tertiary care hospital in Bengaluru, South India. All the isolates were highly resistant to ciprofloxacin. Molecular characterization of these samples performed using Staphylococcal Cassette Chromosome typing and multilocus sequence typing showed that 37.5% of total isolates and 59% of MRSA were sequence type (ST)772 and the rest were other STs. This indicates high prevalence of CA-MRSA in this tertiary care hospital serving the Indian community. Mutations responsible for ciprofloxacin resistance among these isolates in DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB) were analyzed by PCR amplification of specific fragments and sequencing. We found that for ST772 and five other STs present in this collection, single mutation in the gyrA gene, Ser-84→Leu, was sufficient for the high resistance. In vitro generation of ciprofloxacin resistance in two sensitive ST772 isolates by exposure to increasing antibiotic concentrations also resulted in the same single mutation of gyrA. The factors responsible for high ciprofloxacin resistance are varied and are dependent on the genetic background of the isolates and the environment. This is the first report on the mechanism of ciprofloxacin resistance among the most prevalent Indian CA-MRSA.


Introduction
Staphylococcus aureus is an important pathogen in the community and hospital, although 30% of the population is colonized with the organism asymptomatically.Increasing use of systemic antibiotics in the hospitals has resulted in multi drug resistant S. aureus due to selective pressure.The advent of community associated S. aureus into hospitals in the past ten years has resulted in drug resistance in this population too, although the genetic back grounds of community and hospital associated S. aureus are different [1].
Fluoroquinolones (FQs) are broad-spectrum antibiotics effective against gram-positive and -negative organisms, including both methicillin-sensitive and resistant S. aureus (MSSA and MRSA).Ciprofloxacin is the most widely used antimicrobial agent among FQs due to its greater potency.But its use in treatment of S. aureus infections is impaired by the rapid emergence of resistant strains, a feature widely spread among both MRSA and MSSA [2,3].
FQ drugs act by inhibiting the DNA gyrase (gyrA and B) which relieves DNA super coiling and topoisomerase B. CHAKRAKODI ET AL. 134 IV (grlA and B), which separates concatenated DNA strands.Amino acid changes in critical regions of this enzyme-DNA complex reduce the affinity for these drugs.DNA gyrase subunits are common sites of resistant mutations in Gram negative organisms, while topoisomerase IV is most critical in case of Gram positive organisms although there are few exceptions [4].The stepwise emergence of ciprofloxacin resistance in S. aureus is attributed to first grlA mutations conferring low-level resistance, followed by gyrA mutations leading to high-level resistance [5,6].Several efflux pump systems also contribute to FQ resistance in S. aureus [7].
Ciprofloxacin is used commonly in Indian hospitals and reports of widespread resistance exist [8,9].There has been no systematic analysis of ciprofloxacin resistance associated with particular genetic back grounds or diseases [10].This study involves ciprofloxacin resistant MRSA and MSSA isolates, their genetic backgrounds and mutations important in causing the resistance as we have analyzed the clonal complexes present among Indian S. aureus isolates in an earlier study [11].ST772 (single locus variant of ST1) is one of the prominent sequence types present among Indian community associated S. aureus, and there is not much information on resistance to this antibiotic and its mechanism [11][12][13].This work demonstrates that a high percentage of patients in this tertiary care hospital carry CA-MRSA which is highly resistant to ciprofloxacin and a single mutation in gyrA gene is responsible for high resistance in ST772.Mutations among S. aureus isolates from other genetic back grounds are also examined.

Sample Collection
Single sample was collected from each in-patient after admission and cultured for S. aureus within 48 hrs after admission.Forty eight S. aureus isolates were collected over a period of three months (between November 2011 and February 2012) from St. John's Medical College, a tertiary care teaching hospital, Bengaluru, India.Community associated (CA) S. aureus was defined according to Centers for Disease Control and Prevention (CDC) definition for CA-MRSA when patients did not meet any of the following criteria: 1) history of hospitalization, surgery, or dialysis within one year of the SA culture; 2) presence of an indwelling catheter or a percutaneous device at the time of culture.
Hospital had obtained the informed consent from each patient after explaining the purpose of collection.The mean age of the patient was around 40.
Clinical History: Forty percent of patients had superficial skin infections, 29% profound skin infections, 17% pneumonia, 6.3% bone and joint infections, and 8.3% other infections.Major risk factors for infection were diabetes, immuno suppression and burns.

Antibiotic Susceptibility and Minimal Inhibitory Concentration (MIC) Determination
Antibiotic susceptibility testing was performed for erythromycin, gentamicin, amikacin, tetracycline, chloramphenicol and ciprofloxacin by Kirby-Bauer disc diffusion according to the guidelines recommended by the Clinical Laboratory and Standards Institute on Mueller-Hinton agar plates (HIMEDIA, Mumbai, India) at 37˚C.MIC for Ciprofloxacin was determined by the broth dilution method in Mueller-Hinton broth (HIMEDIA) after 24 h of incubation at 37˚C in micro titer plates [15].

Chromosomal DNA Isolation
DNA was isolated from 18 hour grown cultures in BHI broth according to previously published procedures [16].

Molecular Characterization
Staphylococcal cassette chromosome mec (SCCmec) typing was carried out by determining the type of mec and ccr complexes and identification of SCC mec types III, IV and V by an updated multiplex PCR using published procedures and primers [17][18][19].Detection of the Panton Valentine Leukocidin (pvl) gene and accessory gene regulator (agr) typing was carried out by PCR and multiplex PCR respectively [20,21].

Staphylococcal Protein A (spa) Typing and Multi Locus Sequence Typing (MLST)
Spa typing was performed according to the procedure of Shopsin et al. and MLST by Enright et al. [22,23].Pulsed Field Gel Electrophoresis: PFGE was performed using conditions described previously after restriction digestion using SmaI enzyme (Fermentas, India) and CHEF-DRIII (BioRad Laboratories [India] Private Limited, Bengaluru, India) device [16].

Detection of Mutations
Four independent PCRs were carried out to amplify DNA from the quinolone determining region (QRDR) of the gyrA, gyrB, grlA and grlB using the primer sequences published for the four genes [10].PCR products were purified (QiaQuick PCR purification kit; QIAGEN GmbH, Hilden, Germany) and commercially sequenced (Macrogen, Inc, Seoul, South Korea).Wild type sequences and mutations were identified by comparison with the published sequences of ciprofloxacin sensitive S. aureus strains ATCC 12600 and RN4220 for gyrA, gyrB [24] and grlA, grlB [25] respectively.

Generation of in Vitro Ciprofloxacin Resistance in Ciprofloxacin Sensitive ST772 Isolates
Two ciprofloxacin sensitive ST772 isolates with MIC <2.0 µgml −1 were chosen and passaged in Mueller Hinton broth containing increasing concentrations of ciprofloxacin (0.5, 5.0, 50.0 and 500 µg −1 ) growing at each concentration for 48 hrs before increasing to the next.
DNA was made at each step, PCRs were performed for gyrA, and PCR purified products were sequenced.

S. aureus among Admitted Patients
Data collected from interviews and culture results of 48 patients, indicate that about 71% of the admitted patients are colonized with CA and 29% with HA S. aureus according to the criteria given earlier.This is indicative of high rate of colonization among Indian community with S. aureus which could be making an entry in to the hospital.But dividing samples as coming from the community or hospital based on culture results within or after 48 hrs of admission to the hospital under Indian conditions is problematic due to difficulty in gathering accurate data on stay in hospitals, antibiotics taken etc.Most patients would be visiting multiple hospitals and are perhaps not aware of nature of medicines taken.An additional limitation to arrive at conclusions about CA or HA status is the small sample size in this study.

Molecular Characterization
Among 48 S. aureus isolates studied, 43 were ciprofloxacin resistant and 5 were sensitive out of which 40% of the isolates were from superficial skin, 29% from profound skin infections, 17% from pneumonia, 6.3% from bone osteomyelitis/osteitis, and rest from others (eye, septicemia, UTI etc.: 8.3%).

Relationship between Ciprofloxacin and Methicillin Resistance
Several previous studies have indicated that a high percentage of MRSA are resistant to ciprofloxacin compared to MSSA [26].This seems to be true of clinical infections of various organs including the eye although resistance has been reported to be on the rise even among the MSSA [27,28].Among the isolates we have studied, high ciprofloxacin resistance (>512 µg•ml −1 ) is prevalent    among MRSA and MSSA, the number of MSSA isolates studied being lower.High resistance is observed across all isolates with different genetic and clinical backgrounds.Although it is difficult to categorize S. aureus as community or hospital associated among Indian isolates, from the molecular characterization data, it is clear that ciprofloxacin resistance is more prevalent in MRSA carrying SCCmec elements IV and V (ST772, 22, 1208) than III (ST239) indicating S. aureus of community rather than nosocomial origin.Ciprofloxacin resistant MSSA isolates belong to more diverse genetic backgrounds of ST30, 5, 772, 291 etc., also pointing to community origin of the organism.From our data collected in 2003-4 on molecular characterization of S. aureus collected from this and few other Indian hospitals, it was apparent that majority of isolates were MRSA belonging to ST239 carrying SCC mec elements III or IIIa [16].A percentage distribution of 41, 34 and 25 for presence of SCCmec elements V, IV and III respectively, has been published for S. aureus collected in a tertiary care hospital in Mumbai, India, from 2006-9 [12].Our samples collected three months ago have significantly higher proportion of ST772 isolates in this tertiary care hospital.

Mutations
As our isolates belonged to a diverse group of STs, with different genetic back grounds, we studied the mutations in gyrA, gyrB, and grlA, grlB genes that have been shown to play a prominent role in resistance.PCRs were done for the four products using published primers and purified PCR products were sequenced to check for mutations in the four genes as shown in

Number of Mutations
In the last twenty years, S. aureus isolates from different parts of the globe have been tested for mutations causing ciprofloxacin resistance [3].Schmitz et al. had tested isolates from seven different countries and had found that all isolates that did not have grlA mutation were ciprofloxacin susceptible and all isolates which had grlA mutation of Ser-80→Phe and gyrA mutation of Ser-84→Leu or Glu-88→Lys had an MIC of > 4 µg•ml −1 [10].Ng et al. had provided evidence that grlA was the primary target of fluoroquinolones in S. aureus and not gyrA [6].
There was a large study in Japan with 451 isolates looking at gyrA mutations where Ser-84→Leu or Ser-84→ Val showed the highest level of resistance, and Glu-88→ Lys the second highest level of resistance to FQs.The resistance of strains with double mutations was higher than ones with single mutations [29].The same studies have shown increasing MIC to ciprofloxacin with multiple mutations.The MICs reported in most studies were not as high as we are seeing in our isolates.Only one mutation Ser-84→Leu seems sufficient for six STs in our collection to become highly resistant to ciprofloxacin.As ST772 is 37.5% of total number of isolates and 59% of MRSA in our collection, a single amino acid change at Ser-84→Leu seems the only important factor for resistance in this major Indian ST of importance in the Indian hospitals and community.

In Vitro Generation of Ciprofloxacin Resistance in ST772 Isolates
As all our ciprofloxacin resistant isolates had high MIC (>512 µg•ml −1 ), we chose two ciprofloxacin sensitive ST772 isolates, one MSSA and one MRSA, to check mutations in gyrA gene at different concentrations of ciprofloxacin.We generated ciprofloxacin resistance by step wise exposure and examined the change in the gyrA gene at every step.Except for passage with 0.5 µg•ml −1 ciprofloxacin, all other higher concentrations up to 500 µg•ml −1 had the same Ser-84→Leu mutation with no other change in the gyrA gene sequence corroborating the data obtained from ST772 isolates from the collection.We have not checked with other STs as samples were few and we did not have ciprofloxacin sensitive S. aureus isolates.

Conclusion
Most important mutations that have been reported in the literature for ciprofloxacin resistance in S. aureus involve these four genes and our studies indicate that the majority of Indian STs present require only a single mutation in gyrA to acquire high resistance to ciprofloxacin.Few isolates from other STs, 239, 22, 1208, and 9 had multiple mutations and the roles played by these in acquiring the resistance have not been looked into.We have not studied roles played by other factors like efflux proteins in ciprofloxacin resistance in our isolates.As a single mutation is leading to high antibiotic resistance in Indian STs, the genomic background and the environment in which isolates have to survive (high usage of antibiotics in India) might play a very important role.Single mutation in gyrA gene resulting in high resistance to ciprofloxacin may also explain the success of ST772 as a pathogen similar to ST22 isolates with SNPs resulting in ciprofloxacin resistance [30].

Figure 1 .
Figure 1.PFGE patterns and dendrogram of ciprofloxacin resistant and sensitive isolates belonging to different genetic backgrounds.

Figure 2 .
Figure 2. ClustalW alignment of gyrA gene sequences of ciprofloxacin passaged ST772 MSSA and MRSA with ciprofloxacin sensitive control ATCC12600.

Table 1
cin.Majority of isolates were resistant to erythromycin while the other antibiotics were resistant to varying degrees.Out of 19 isolates from superficial skin infections, 17 were ciprofloxacin resistant and 2 were sensitive.Out of 14 isolates from profound skin infections, 12 were

Table 3
. A common mutation in gyrA serine 84 to leucine was found in S. aureus isolates of all genetic back grounds.STs 772, 30, 291, 5, 7, and 672 had only this mutation in gyrA whileOPEN ACCESS AiM

Table 3 . Mutations in gyrA, gyrB, grlA and grlB genes among S. aureus Isolates of Different Genetic Backgrounds.
tive as well as resistant ST772 isolates did not have any change in bases or amino acids in grlB (Figure2).